Inhibitory effects of Serjania erecta on the development of Chrysodeixis includens

The soybean looper, Chrysodeixis includens, is a primary soybean pest that reduces crop productivity. This work examined control of C. includens populations with methanolic extract of Serjania erecta, a native Cerrado plant, while minimizing risks to pollinators, natural enemies and the environment. Serjania erecta specimens were collected, identified, and subjected to methanol extraction. Bioassays were performed using newly hatched and second-instar caterpillars and different extract concentrations on the diet surface to obtain IC50 values. Two replicates, containing 10 caterpillars, were established in triplicate. The IC50 values were 4.15 and 6.24 mg of extract mL−1 for first-instar and second-instar caterpillars, respectively. These growth inhibition results informed the extract concentrations assessed in subsequent development inhibition assays, in which the pupal weight was higher under the control than under the treatments. Extract treatments increased the duration of the larval, pupal and total development. The potential of different concentrations of S. erecta extract to inhibit the enzymes carboxylesterases was also evaluated. Carboxylesterases activity decreased by 41.96 and 43.43% at 7.8 and 15.6 μg mL−1 extract, respectively. At 31.3 μg mL−1 extract, enzymatic activity was not detected. Overall, S. erecta leaf methanolic extract showed inhibitory potential against carboxylesterases.

www.nature.com/scientificreports/ affect insect resistance to insecticides 16 . In addition to enzyme inhibition, other effects of botanical insecticides on insects include morphological deformities on S. litura 8 , antinutrient action on B. longissima 9 , mortality of Helicoverpa armigera, S. litura, B. longissima and Sitophilus zeamais 6,8,9,17 , repellence against S. zeamais and H. armigera 17,18 , inhibition of growth on H. armigera 6 , ovicide against Rhodnius prolixus 19 , and delayed development of S. litura and B. longissima 8,9 . At least 12,000 higher plants are estimated to occur in the Brazilian Cerrado savanna 20 . Serjania erecta, known as the five-leaf vine, is native to the Cerrado and produces numerous secondary metabolites, such as saponins, flavonoids, tannins, and terpenes 21 , which are components contributing to the high insecticidal activity observed in plants of the Sapindaceae 22 .
One of the most effective ways to minimize the loss of biodiversity is the development of conservation programs and the sustainable use of biological resources 23 . Given the biotechnological potential of S. erecta, it is important to guarantee its conservation. This plant may provide compounds that control C. includens, contributing to a reduction in the application of synthetic insecticides. The present study investigated the weight of newly hatched C. includens caterpillars and second-instars, the development of the immature stages following exposure to the methanolic extract of S. erecta leaves, and the effects of this extract on carboxylesterases enzymes.

Materials and methods
Insect source. The initial population of C. includens was established from pupae reared in the laboratory and provided by the ESALQ-USP Entomology Laboratory. As this study did not use caterpillars collected from natural populations, however, all necessary licenses and permissions were obtained for the development of the study, including a license to collect (SISBio -769521) and permission for in vitro cultivation (ordinance 265 of 07/22/19 of the IFGoiano campus Rio Verde). The caterpillars were raised in the Entomology Laboratory of the Rio Verde campus of the Goiano Federal Institute at 25 ± 2 °C, a relative humidity of 60 ± 10%, and a 14-h photophase.
Insect rearing. The caterpillars were raised on an artificial diet based on white bean 24 . The adults were housed in enclosures made from PVC tubes (20 cm in length and 15 cm in diameter) on a 20-cm-diameter plastic base plate and covered with organza-type fabric held in place with an elastic band. The enclosures were lined with sheets of A4 paper as a substrate for egg laying. A 20 mL container with a wad of cotton soaked in a 10% honey solution was added to each enclosure. The enclosures were cleaned and provisioned daily when any eggs were removed. Once hatched, the caterpillars were maintained in pairs in 50 mL plastic containers, with a portion of the feed (1.5 cm × 1.5 cm). These containers and the feed were changed twice a week. The pupae were removed and housed in 500 mL plastic containers until emergence of the adults, which were transferred to the enclosures described above. The C. includens populations were maintained in isolation at a temperature of 25 ± 2 °C and relative humidity of 60 ± 10% with 14-hourphotophase. Acquisition of the extract. The leaves of S. erecta were dried for 5 days at 40 °C in an air-circulated Marconi MA 035 stove until reaching a constant weight. The samples were then triturated to a powder in a Willey Tecnal TE 680 knife mill. The extracts were obtained from this powder, following the methodology proposed by Fernandes et al. 21 . Thus, 1 L of methyl alcohol was added per 200 g of powder. This mixture was maintained in sealed Erlenmeyer flasks for 24 h and then filtered. The filtered samples were processed in a Tecnal TE-210 rotary evaporator at 65 °C, a temperature close to the boiling point of the solvent. The solvent recovered at the end of the process was added to the pellet resulting from the initial extraction, and the whole procedure was repeated twice. The extract was then transferred to a lidded glass container to be frozen and lyophilized in a Liotop L108. The lyophilized product was stored at ambient temperature until the bioassays.
Assays of the inhibition of caterpillar growth. Newly hatched and second instar C. includens caterpillars were exposed to five different concentrations of the extract: 1.25 mg L −1 , 2.5 mg L −1 , 5.0 mg L −1 , 10.0 mg L −1 , and 20.0 mg L −1 (v/v). The control treatment was distilled water with 2.5% methyl alcohol (v/v). All bioassays were run twice in triplicate. For each concentration, 300 μL of the extract solution was applied to the surface of 10 mL of the artificial diet, and the diet was placed in 100 mL plastic containers. The solution was then spread over the surface of the feed with circular movements. These containers were left without lids for two hours until all the excess moisture had dried off. Subsequently, 10 newly hatched caterpillars or second instars were transferred to each container, which was then closed and labeled with the age of the insect and the concentration of the extract. After 7 days, the surviving caterpillars were weighed on an analytical balance, and the mean weight of the caterpillars was obtained for each concentration of the extract. The mortality of the caterpillars was also quantified.

Assays of the inhibition of caterpillar development.
The results of the caterpillar growth inhibition assays were used to define the concentrations of S. erecta leaf extract to be evaluated in the caterpillar develop- www.nature.com/scientificreports/ ment inhibition assays, i.e., 10 mg L −1 and 20 mg L −1 of extract. The assays were based on similar procedures, with the methanolic extract of S. erecta being diluted in distilled water containing 2.5% (v/v) methyl alcohol. In this case, however, five repetitions were conducted per concentration, in addition to the control, which was distilled water containing 2.5% (v/v) methyl alcohol. After 7 days, the caterpillars were removed from the containers with the extract applied to the surface of the feed. They were then placed in individual containers and fed continuously on the artificial diet with no added extract. The duration of the larval and pupal stages and the timing of the emergence of the adults were determined through daily monitoring of the containers. The pupae were sexed to verify the sex ratio.  The data on the weight of the pupae, the duration of the larval and pupal phases, total development, and viability of the immature moths were first evaluated for normality using the Kolmogorov test (SAS Proc Univariate) and for homogeneity using Bartlett's test and subsequently submitted to analysis of variance. Significant differences between the means were compared by the Tukey test at 5% probability. Similar tests were applied to the enzyme activity data.

Enzyme inhibition assays.
Ethics Statement. This study did not use any vertebrate animal.

Results and discussion
To test the hypothesis that the methanolic extract of S. erecta leaves influences the development of C. includens caterpillars, these animals were fed diets with or without the extract for 7 days. Caterpillars of different ages were then weighed to determine the potential inhibition of growth. Development inhibition trials were then run using recently hatched caterpillars, which were presented with diets with/without the extract for 7 days, after which they were given a normal diet without the extract. During these trials, the mean duration of the larval and pupal stages and the full developmental period were recorded, and the viability of the immature stages was determined.
The body mass data were adjusted to a logistic nonlinear regression model with three parameters to estimate the inhibition of growth, IC 50 , as shown in Table 1. The IC 50 value was significantly higher for the second-instars (t = 1.0021 14,25 ; P = 0.0444) than for the newly hatched caterpillars. Based on the ratio of the IC 50 values recorded in the two groups, the extract was 1.5 times more potent to newly hatched caterpillars than to the second-instars.
The methanolic leaf extract of S. erecta was 1.5 times more potent to newly hatched C. includens caterpillars than to older individuals, although biologically, this difference may not have any major implications. These www.nature.com/scientificreports/ findings are consistent with existing data, which indicate higher mortality in first-instar C. includens caterpillars following exposure to the unrefined extract of Annona mucosa seeds than in third-and fifth-instar caterpillars 26 .
Other studies have attributed the greater susceptibility of younger caterpillars to the reduced effectiveness of detoxifying intestinal enzymes in these herbivorous lepidopterans 27 . In the present study, the 10 mg mL −1 and 20 mg mL −1 concentrations of the extract reduced the mass of newly hatched caterpillars by 92% and 93%, respectively. However, no significant difference was recorded in the mortality of the control and treatment groups ( Table 2). The 10 mg mL −1 and 20 mg mL −1 extract concentrations delayed larval, pupal and total development significantly in comparison with the control ( Table 2). The body mass also decreased significantly in both treatments in comparison with the control (Table 2), although viability was not significantly different in any of the analyses. The development of the caterpillars returned to normal once the individuals were transferred to a normal diet (without the extract), although their delayed developm ent and reduced weight may make them more susceptible to other factors, such as predators and sunlight. The sublethal effect of underdeveloped wings may be associated with a reduction in longevity, as well as reduced reproductive success, due to its influence on other aspects of the life of the insect, such as alterations of its immune system and behavior 4 . Table 1. Parameters of the nonlinear logistic regression plotted to estimate the patterns of weight reduction in newly hatched and second-instar Chrysodeixis includens caterpillars exposed to different concentrations of methanolic extract of Serjania erecta leaves. 1 a corresponds to the difference between the minimum and maximum points of the curve; 2 b corresponds to the curve slope; 3 SEM corresponds to the standard error of the mean.  Figure 1. Caterpillars of Chrysodeixis includens exposed to the control diet and a diet containing the methanolic extract of Serjania erecta leaves at a concentration of 20 mg mL −1 (a) and normal Chrysodeixis includens adults and morphologically deformed individuals (right) exposed to methanolic extract (b). www.nature.com/scientificreports/ The delayed development and weight loss observed in C. includens may have been related to the phenolic compounds found in the S. erecta extract, including kaempferol 28 , saponins 29 , and apigenins 28 . Phenolic compounds may cause a significant reduction in the survival and growth of insects due to the inactivation of digestive enzymes 30 . Caterpillars of Pieris brassicae (Lepidoptera) exposed to a diet treated with kaempferol suffered a significant reduction in body mass 31 . Extracts of Sapindus mukorossi (Sapindaceae), which include saponins, caused a reduction in the feeding of Thysanoplusia orichalcea (Lepidoptera) and inhibited both larval and pupal development 32 , which may have been related to the hydrolysis of the saponins into aglycons, which are liberated by the glycosylases found in the intestine of the insect 32,33 . Cipollini et al. 34 showed that purified apigenin reduced feeding in Spodoptera exigua (LepidopteraNoctuidae) caterpillars and may be capable of interrupting the molting cycle 35 .
When verifying the enzymatic activity in the presence of 500 µM of substrate, at concentrations of 7.8 and 15.6 µg mL −1 of extract, the activity of carboxyl-esterases decreased by 41.96 and 43.43%, respectively (Fig. 2). At a concentration of 31.3 µg mL −1 of S. erecta leaf methanolic extract, it was not possible to detect enzymatic activity.
The inhibitory effect of plant extracts has been known for a long time 36 . In these works, secondary metabolites such as saponins, flavonoids, tannins and terpenes seem to act in different ways on the activity of enzymes important for the metabolism of various organisms 8,37,38 .
Regarding S. erecta, there are few reports in the literature regarding the inhibitory activity of its extracts. Broggini et al. 29 showed the inhibitory effect of crude extracts of this species using thin layer chromatography. In this work, the flavonoid fraction of this extract was shown to be more selective for inhibition of acetylcholinesterase than for that of butyrylcholinesterase, while the saponin fraction showed greater inhibition of butyrylcholinesterase than of acetylcholinesterase 29 . Thus, the presence of metabolites in the extract of this plant, at a concentration of 31.3 µg mL −1 , is high enough to totally inhibit the activity of carboxyl-esterases.
In the present work, to characterize the kinetic behavior of these enzymes in the absence and presence of the methanolic extract of S. erecta leaves, steady-state kinetics experiments were carried out, following the Michaelis-Menten model (Fig. 3).
Analyzing the results according to the Michaelis-Menten model, it is possible to obtain the maximum velocity of the enzyme (Vmax), as well as its affinity constant (Michaelis constant, KM), as shown in Table 3.
In the presence of the extracts, the enzymatic activity is disturbed to the point of not allowing a good adjustment of the experimental data to the theoretical curve, reflected in high errors, mainly with regard to the determination of KM and R 2 . However, it can be noted that in the presence of the extracts, a decrease in Vmax and, at least www.nature.com/scientificreports/ at the lower concentration of extract, a maintenance of the KM values could indicate a mixed-type inhibition. This can be seen in the graph of reciprocal doubles, shown in Fig. 4, which shows typical mixed inhibition behavior. Overall, the results of the present study indicate that the insecticidal effects of the methanolic extract of S. erecta leaves on C. includens are probably related to the inhibition of the enzymes of the digestive tract, which reduces the absorption of nutrients, thereby impacting the development of the caterpillars. However, we cannot  Table 3. Vmax (UA/s) and KM (μM) of carboxylesterases extracted from intestines of fourth-instar larvae of Chrysodeixis includens in the absence and presence of 7.8 and 15.6 μg mL −1 of methanolic extract (20 mM sodium phosphate buffer, pH 7.0, T = 22 °C) from Serjania erecta leaves. 1 Values after " ± " are the standard error of the mean.